Method for screening white blood cells

ABSTRACT

A method and apparatus for automatically and rapidly, retrieving, counting and/or analyzing at least one selected white blood cell population and/or subset thereof of an aged whole blood sample or portion thereof. A volume of a biological sample containing the white blood cells is prepared and at least one reactant specific or preferential at least to some selected biological cells is introduced thereto and rapidly mixed for a short period of time. The total white blood cell populations and lymphocyte population are first electronically counted and then the neutrophil population including the aged neutrophil population is removed from the same or a second sample portion. The opacity and/or volume parameter of the cells then can be modified and the mixture then again is counted and analyzed in one or more steps to obtain the desired white blood cell population analysis. 
     The reactant can include or be a lyse or a monoclonal antibody bound to microspheres, which will bind to specific ones of the cells or a combination of lyse and microspheres with antibody bound thereto. The microspheres can be magnetic and the bound cells can be magnetically removed for retrieving and analyzing the remaining blood cell population.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of co-pending application,U.S. Ser. No. 587,646, filed Sept. 20, 1990, which in turn is acontinuation of U.S. Ser. No. 025,345, filed Mar. 13, 1987 nowabandoned, the disclosures of which are incorporated herein byreference.

BACKGROUND OF THE INVENTION

This invention relates generally to a method and apparatus for screeningcells in aged samples. More particularly, the invention is directed toan analysis of the aged cells by utilizing microspheres having specificmonoclonal antibodies bound thereto to remove one cell population and toobtain a multi-part white blood cell population differential.

This invention relates generally to an automated analyzer and methods ofusing same for screening biological cells or formed bodies for theenumeration of populations which express selected characteristics forresearch, diagnostic, medical or industrial purposes. More particularly,the automated analyzers and methods embodying the invention enablemultiple part classifications of cells and formed bodies, functionalphenotyping of cells and formed bodies, typing of leukemic, lymphoma andsolid tumor cells, among others, using a unique combination ofelectronic and optical technology and the specificity of selectivebiological molecules, such as antibodies, for such screening andselective enumeration of the cells and formed bodies.

Automation of routine complete blood cell (CBC) analysis of humanperipheral blood by an automated blood cell counter was successfullyachieved by the COULTER COUNTER (Registered Trademark) Model A ofCoulter Electronics, Inc. of Hialeah, Fla. The electronic particlesensing system principle of that instrument is disclosed in U.S. Pat.No. 2,656,508 issued Oct. 20, 1953 to Wallace H. Coulter. The use ofoptical sensing means or lasers, which can be troublesome and expensiveare avoided by particle analyzing instrumentation solely operated onthis Coulter electronic sensing principle.

This Coulter sensing principle was developed and expanded into moresophisticated instrumentation such as the COULTER COUNTER (RegisteredTrademark) Model S types of instruments which enabled CBC parameters,absolute cell counts, platelet count and morphology, red blood cell(RBC) morphology, interpretation of normal and abnormal blood specimensby specific computer programs.

The Coulter electronic particle sensing principle employs an aperturesensing circuit using a direct current (DC) aperture supply. Suchparticle sensors are simple in structure, extremely rugged and reliableas attested to by the substantially universal acceptance of the COULTERCOUNTER (Registered Trademark) automated analyzer in clinicallaboratories in the United States and throughout the rest of the World.An improvement in this basic aperture sensing circuit was disclosed inU.S. Pat. No. 3,502,974 issued in 1970 to Wallace Coulter and WalterHogg. In addition to the standard direct current aperture supply, a highfrequency aperture current was applied which enabled the sensing of anadditional parameter for classification purposes. The high frequencyaperture current produced a signal

the function of the blood cell's internal conductivity as well as itsvolume. The signal produced simultaneously by the direct currentaperture circuit is a conventional DC amplitude signal which provides anindication primarily of cell volume. The radio frequency amplitude isdivided by the direct current pulse amplitude employing a high speeddivider circuit to obtain a quotient which is a function of cell volumeand internal resistance, conveniently referred to as "opacity". Thisprinciple is further described in U.S. Pat. No. 3,502,973 also issued toWallace Coulter and Walter Hogg, in 1970. This parameter hasapplicability in cell classification systems. Either a single or a pairof separate apertures could be utilized for this purpose.

Classification of different populations is accomplished by collating thedata of the signal pairs as they are produced; one, a measure ofparticle volume and the other a measure of cell internal resistivity oropacity. A convenient form of presenting this data is by two-dimensionalplots referred to as scatterplots or scattergrams. Such plots are welldescribed in Flow Cytometry and Sorting, page 371; edited by MelamedMelaney, and Medelsohn, 1979, John Wiley & Sons, NY, N.Y.

FIG. 5A is one example of a data plot of a sample of normal blood. Eachdot represents an individual cell. The height above the baselinerepresents the relative volume of the cell. The distance of the dot tothe right of the vertical baseline represents the relative opacity. Aplot of normal white blood cells (WBC) (with the red blood cellsremoved) shows three clusters of dots representing three distinctpopulations which are a consequence of their intrinsic differences insize and internal composition. If desired, with suitable circuitry,these populations can be enumerated to obtain the numbers of each. Thecells are classified on the basis of these inherent differences.

Initial applications of the Coulter electronic particle sensingprinciple was to perform red blood cell counts and then, moresophisticated determinations of other red blood cell parameters. Byremoving red blood cells from whole peripheral blood, analysis of thewhite blood cell populations could be undertaken so long as the redblood cell removal did not significantly impair properties of theremaining white blood cell populations sought to be measured. Red bloodcell lysing reagents were developed for this purpose which, thoughuseful and widely applied, were not entirely satisfactory in allrespects for subsequent white blood cell determinations.

Previous methods of flow analysis of leukocytes using DC volume alone orlight scatter at various angles have shown three clusters of leukocytescorresponding to lymphocytes, monocytes and granulocytes which includedthe neutrophil, basophil and eosinophil populations. A rough but usefulestimation of eosinophil concentration can be made on some samples. Thefifth major population is relatively too small for this approach. Theeosinophils also have been observed as a distinct cluster using specialfluorescence techniques.

These fluorescent techniques were utilized in flow cytometry instrumentssuch as the EPICS (Registered Trademark) flow cytometer available fromthe Coulter Corporation. Such instruments employed the principle ofcells moving in a columnar stream bounded by a sheath flow such thatcells lined up in single file and passed individually through a laserbeam. Light scatter and/or fluorescence signals from the cells were thenutilized in classifying cell populations. Staining cells with absorptiveor fluorescent dyes made additional cell population classificationspossible. The development of instrumentation and fluorochromes forautomated multiparameter analysis is further described by R. C. Leif, etal. in Clinical Chemistry, Vol. 23, pp. 1492-98 (1977). Thesedevelopments expanded the number of simultaneous populationclassifications of leukocytes to four, namely lymphocytes, monocytes,eosinophils and "granulocytes" (neutrophils and basophils).

A more recent analytical hematology instrument has utilized lightscattering techniques together with peroxidase enzyme staining(absorptive dye) of cells to produce a five part leukocyte differential.Moreover, dyes in combination with specific reacting biologicalmolecules, such as monoclonal antibodies, have increased the number ofleukocyte classifications possible to include functional subdivisions.

An improved single automated instrument and methods of using the same isdisclosed in parent application U.S Ser. No. 587,646, filed Sept. 20,1990, entitled AUTOMATED ANALYZER AND METHOD FOR SCREENING CELLS ORFORMED BODIES FOR ENUMERATION OF POPULATIONS EXPRESSING SELECTEDCHARACTERISTICS, which is a continuation of U.S. Ser. No. 025,345, filedMar. 13, 1987 of the same title. This application combines theapplication of electronic sensing aperture principles, the specificityof selected biological molecules for identifying and/or enumeratingdefined populations of cells or formed bodies and microscopic particletechnology. The automated analyzer can be used together with a speciallysing reagent and/or antibodies coupled to microscopic microspheres orsupports of varying composition.

A second application, U.S. Ser. No. 285,856, filed Dec. 16, 1988,entitled METHOD AND APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITHPOPULATIONS EXPRESSING SELECTED CHARACTERISTICS, discloses the screeningof direct subsets from whole blood samples or portions thereof.

A third application, U.S. Ser. No. 339,156, filed Apr. 14, 1989,entitled METHOD AN APPARATUS FOR SCREENING CELLS OR FORMED BODIES WITHPOPULATIONS EXPRESSING SELECTED CHARACTERISTICS UTILIZING AT LEAST ONESENSING PARAMETER, discloses multipart or five part white blood celldifferentials, lymphocyte subsets and overlapping determinationsperformed from a whole blood sample or from a sample with the red bloodcells and/or populations of the white blood cells removed by eliminationof populations and/or subsets thereof with one or more light orelectronic parameters.

A fourth application, U.S. Ser. No. 07/525,231, filed May 17, 1990,entitled METHOD AND APPARATUS FOR SCREENING OBSCURED OR PARTIALLYOBSCURED CELLS, discloses an analysis of obscured cells by utilizingmicrospheres having specific monoclonal antibodies bound thereto to movethe sensed characteristics of the obscured cells from one cellpopulation area on a scattergram to another area. Each of the four abovereferenced applications is incorporated herein by reference.

An improved analytical hematology instrument and methods of utilizingthe same are disclosed in U.S. Ser. No. 025,442 filed Mar. 13, 1987 andcontinuing U.S. Ser. No. 129,954 filed Dec. 4, 1987, both entitledMULTI-PART DIFFERENTIAL ANALYZING APPARATUS UTILIZING LIGHT SCATTERTECHNIQUES and are incorporated herein by reference. This so called"VCS" hematology instrument utilizes light scattering and electronicsensing techniques to obtain a multi-part differentiation of theleukocyte (L) WBC population. This hematology instrument, however, doesnot perform differentiation of L subsets, since such subsets areobscured in the L population.

Selectively attaching microscopic particles to each cell of a cellpopulation makes possible the modification of the parameter(s)responsible for the original location of at least one of thepopulations. The addition of a plurality of microscopic particles toeach cell of selected target populations where this addition affects themeasured volume and/or opacity results in shifting the location of thedots in the scattergram representing a population.

Antibodies of known specificity are employed in coating microscopicparticles. This coating gives the particle the capacity to selectivelyattach to certain cells which express the antigen the antibody isspecific for. These coated or tagged cells are a combination ofparticles and a cell which behave like a new entity. Their parameters ofopacity, volume, or both opacity and volume may be considered torepresent the sum of the effects of both the cell and the particles onthe signals obtained. If the characteristics of the components aredifferent, the new entity will move to a new position on a scattergramin accordance with the net effect. The new location, in contrast withthe former position of the cell alone, should allow a classification ofsuch new entity or group of new entities. If the particles attached tothe cells are magnetic, then, of course, according to current practice,the new entities can be captured by the use of a magnet. If mixedrapidly, unexpected results including complete capture of a populationwithout adversely affecting the properties of the cells under studyoccur.

Only three distinct populations of cells can be readily identified andenumerated from a blood sample by utilizing their inherent and uniqueproperties of DC volume and opacity parameters heretofore stated.Additional steps such as improved lysing systems, must be taken toenable the detection and enumeration of more populations. Of course,these additional populations represent subpopulations of the three basicones referred to as lymphocytes, monocytes and granulocytes. The stepsperformed in accordance with the above referenced applicationsdemonstrate how subpopulations of these basic three populations areobtained.

Employing such simple aperture sensing techniques in combination withtwo or more biological particles, one can produce a unique and newposition of the dot cluster representing a given population. Thisselective movement of populations on the dot plot or scattergram isreproducible and can be used to classify a population separate from thebasic three populations.

The original and inherent combination of DC volume and opacity sensingtechniques can be modified through the attachment of microscopicparticles to selected individual cells. The selectivity is given theparticles by the nature or specificity of the biological molecules,antibodies among others, employed as the coating on the particlesurfaces. A population of cells alone, having no particles on theirsurface, may occupy a dot plot position no different from otherpopulations or subpopulations, and, henceforth, not be distinguishablefrom one another. The addition of particles having a selectiveattraction to a specific population of cells which one seeks toidentify, enumerate, and study is possible using this approach. Theselective addition of a sufficient mass of selective particles to adistinct population of interest results in the shifting of thatpopulation's dot plot location as a result of the new and uniquecombination of mass, volume and opacity of each cell.

Although the term "reactant" has been utilized in some of the aboveapplications to define lysing agents and monoclonal antibodies,reactants can include various agents which detect and react with one ormore specific molecules which are on the surface of a cell. Someexamples are given below:

    ______________________________________                                        Reactant         Specific Molecule                                            ______________________________________                                        Antibody         Antigen                                                      Drug             Drug Receptor                                                Hormone          Hormone Receptor                                             Growth Factor    Growth Factor Receptor                                       ______________________________________                                    

The reactants couple or bind to the specific molecule(s) on the cells.These reactants do form part of a chemical reaction; however, thereactants are not necessarily chemically altered.

One of the most valuable features of this invention is that it employsthe simple rugged Coulter sensing operation. It is stable and does notrequire the complexity and expense of complex optical systems. Thecircuitry required for the addition of the RF generator and detector iseconomical, compact and reliable. A single aperture is all that isrequired, but the addition of a second or even a third aperture canenable a greater sample throughput rate economically.

SUMMARY OF THE INVENTION

The invention provides a method and apparatus for performing screeningof cells in an aged whole blood sample or portion thereof to obtain amulti-part white blood cell population differential. The total whiteblood cell populations and lymphocyte population are firstelectronically counted and then the neutrophil population including theaged neutrophil population is removed from the same or a second sampleportion. The remaining white blood cell population of monocytes,basophils, lymphocytes and eosinophils including the aged eosinophilsthen are electronically counted and the two counts are compared toobtain a five-part white blood cell differential.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1-13 describe the embodiments disclosed in the parent applicationSer. No. 587,646;

FIG. 1 is a schematic block diagram of one cell population analyzerembodiment of the parent application;

FIG. 2 is a schematic block diagram of a second analyzer embodiment ofthe parent application;

FIG. 3 is one specific analyzer embodiment of the parent applicationcorresponding to FIGS. 1 and 2;

FIG. 4 is a schematic block diagram of another analyzer embodiment ofthe parent application;

FIGS. 5A and 5B are a scattergram of one set of results utilizing aprototype analyzer system similar to that illustrated with respect toFIGS. 2 and 3;

FIG. 6 is a schematic block diagram of a further analyzer embodiment ofthe parent application;

FIG. 7 is a schematic block diagram of a still further analyzerembodiment of the parent application;

FIGS. 8A and 8B, 9A and 9B, 10A and 10B and 11A and 11B are ascattergram of one set of results utilizing a prototype analyzer systemsimilar to that illustrated with respect to FIGS. 6 and 7;

FIG. 12 is a schematic block diagram of a yet still further analyzerembodiment of the parent application;

FlG. 13 is a scattergram of one set of results utilizing a prototypeanalyzer system similar to that illustrated with respect to FIG. 12;

FIGS. 14 and 15 describe the embodiments disclosed in the presentinvention;

FIGS. 14A-C are scattergrams of a 24-hour aged sample; and

FIGS. 15A-C are scattergrams of a 36-hour aged sample.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

FIGS. 1-13 describe the embodiments of the parent application, Ser. No.587,646.

Referring to FIG. 1, a first embodiment of a cell population analyzingmethod and apparatus of the parent application, Ser. No. 587,646, isdesignated generally by the reference numeral 10. The analyzer 10includes a biological sample 12 which contains at least a first set ofviable biological cells (not illustrated), such as in or from a wholeblood sample. The cells of the biological sample 12 are to be involvedin a biological reaction in a quantitative and/or qualitativedetermination or analysis. The sample 12 can include a buffer into whichthe cells are added.

The sample 12 is combined via a line 14 with at least one reactant 16via a line 18. The red blood cells (RBC) then are removed from themixture by a functionally designated RBC removing station 20. The RBC'scan be removed from the mixture by the station 20 in a number of ways.The RBC's can be lysed by a lyse in the reactant 16. One suchpreferential lyse and a quench which can be utilized therewith isdisclosed in Ser. No. 130,911, filed Dec. 10, 1987, entitled METHOD ANDREAGENT SYSTEM FOR ISOLATION, IDENTIFICATION AND/OR ANALYSIS OFLEUKOCYTES FROM WHOLE BLOOD SAMPLES, which is a continuation-in-part ofSer. No. 025,303, filed Mar. 13, 1987, of the same title, which areincorporated herein by reference. The reactant 16 can be or include aplurality of magnetic microspheres with an antibody specific to theRBC's bound to the microspheres (not illustrated). In this example, theparticular red blood cell specific antibody utilized is disclosed inU.S. Pat. No. 4,752,563, entitled MONOCLONAL ANTIBODY FOR RECOVERY OFLEUKOCYTES IN HUMAN PERIPHERAL BLOOD AND METHOD OF RECOVERY EMPLOYINGSAID MONOCLONAL ANTIBODY, which is incorporated herein by reference. Thereactant 16 also can include a buffer in addition to or in place of thesample buffer. The reactant 16 further can be a combination of thepreferential RBC lyse and the RBC specific microspheres.

Once the RBC's substantially are removed from the mixture, a portion ofthe mixture is fed into a white blood cell (WBC) analyzer 22 via a line24. The WBC analyzer 22 at least counts the number of WBC's in themixture. The WBC analyzer 22 also can measure one or more volume oropacity parameters of the WBC's. The results from the analyzer 22 arefed to a comparator 26 via a line 28.

A second portion of the RBC deleted mixture is fed to a WBC subsetsubtracting station 30 via a line 32. The WBC's can be subtracted fromthe mixture in a number of ways. Microspheres with a monoclonal antibodyspecific to one of the WBC subsets bound thereto can be added to themixture. Non-magnetic or magnetic microspheres can be bound to the WBC'sto change or shift the resultant opacity or volume parameters of thecells. Magnetic microspheres also can be bound to the WBC's which thencan be removed from the mixture by a magnetic field.

The mixture with the WBC subset population removed or with one or moreparameters changed then is fed to a WBC subset analyzer 34 via a line36. The analyzer 34 can be identical to the analyzer 22. The results ofthe analyzer 34 are then fed to the comparator 26 via a line 38. Thecomparator 26 then can compare the WBC results from the analyzer 22 withthe modified results from the analyzer 34 to determine at least onecharacteristic of the selected white blood cell population, such as thenumber of cells in a particular range.

Referring to FIG. 2, a second embodiment of a cell population analyzingmethod and apparatus embodying the parent application is designatedgenerally by the reference numeral 40. The analyzer 40 includes abiological sample 42 which again contains at least a first set of viablebiological cells (not illustrated), such as in or from a whole bloodsample. The cells of the biological sample 42 are to be involved in abiological reaction in a quantitative and/or qualitative determinationor analysis. The sample 42 again can include a buffer into which thecells are added.

The sample 42 is combined via a line 44 with at least one reactant 46via a line 48. In the analyzer 40, RBC's are removed from the mixtureand simultaneously at least one characteristic of at least one WBCsubset is changed or shifted by a functionally designated RBC removingand WBC shifting station 50. As stated above, the RBC's can be removedfrom the mixture by the station in a number of ways, previouslyenumerated with respect to the station 20. Simultaneously, in the samemixture portion, the WBC's are bound to, generally non-magnetic,microspheres to change or shift the resultant opacity and/or volumeparameters of the cells.

The mixture with the RBC's removed and the WBC subset population shiftedthen is fed to an analyzer 52 via a line 54. The analyzer 52 can besubstantially identical to the analyzer 22. The analyzer 40 thusprovides a fast, direct analysis of at least one characteristic of aselected WBC population or WBC subset.

One specific embodiment of an analyzer instrument embodying the parentapplication and which can accomplish the analyzing methods of the firstand second analyzers 10 and 40, is designated generally by the referencenumeral 56 in FIG. 3.

In the instrument 56, only one specific enumeration is illustrated,which can be varied in almost endless detail in accordance with theprinciples of the parent application. Further, the instrument 56 isshown in generally functional detail and the specific embodiments can bestructurally implemented in many known ways.

The instrument 56 includes an aspirator pumping mechanism 58 which isutilized to draw the biological sample of interest, for example thesample 12 or 42 into the instrument 56. The aspirator 58 is coupled viaa line 60 to a sampling valve 62, which can be coupled to a sample probe63. A diluent pump 64 can include a diluent or buffer solution and alsois coupled to the valve 62 via a line 66. The valve 62 and the pump 58can aspirate the biological sample 12 or 42 along with the diluent viathe pump 64 when appropriate.

The reactant mixture or the biological sample itself, then is fed via adischarge line 68 into a mixing apparatus 70. The mixer 70 includes amixing chamber 72 into which the sample or reactant is fed. The RBC'sthen will be lysed by a lyse from a lyse pump 73 fed into the chamber 72via a line 74. At this point the operation of the analyzer 10 and 40differ and hence will be described separately.

In the case of the analyzer 10, if the RBC's have been lysed by the lysefrom the pump 73, then when the reaction is completed a quench or fix issupplied from a station 75 via a line 76. The reaction can be assistedby mixing the lyse and the sample in the chamber 72 as illustratedfunctionally at 78.

Specific details of an appropriate mixing apparatus 70, which can beutilized herein are disclosed in Ser. No. 517,309, filed May 1990, whichis a continuation of Ser. No. 025,337, filed Mar. 13, 1987, entitledMETHOD AND APPARATUS FOR RAPID MIXING OF SMALL VOLUMES FOR ENHANCINGBIOLOGICAL REACTIONS, which are incorporated herein by reference. Byutilizing the mixer 70 the reactions are greatly enhanced in speedwithout significantly damaging the properties of interest of the cells,such as, can occur by raising the reaction temperature. Further, thereactions generally are completed in significantly less than a minute,generally on the order of fifteen seconds or less. This allows a rapidanalysis of the automatic high volume analyzer instrument 56.

The quenched reactant with the RBC's removed by the lyse (as from thestation 20) then is fed via a line 80 to a holding chamber 82, which inthis case will hold a second portion of the mixture. A first portion ofthe mixture will be fed from the chamber 82 via a line 84 to a WBCanalyzer 86 (i.e., analyzer 22). The analyzer 86 can be of many physicaltypes in accordance with the counting and sizing techniques described byWallace H. Coulter in U.S. Pat. No. 2,656,508 and embodied in thenumerous commercial blood cell counters of the assignee, CoulterElectronics, Inc.

The analyzer 86, in general, includes a flow sensor or sensing chamber88. The chamber 88 includes a transducer 90 which has an aperture 92therethrough. The chamber 88 includes a first portion 94 which has afirst electrode 96 in contact with the fluid therein.

The chamber portion 94 and the electrode 96 communicate through theaperture 92 with a second chamber portion 98 having a second electrode100 therein. The electrodes 96 and 100 are coupled via reactive leads102 and 104 to an RF/DC source and sensing circuit log. The circuit 106couples both a DC, or low frequency current or signal, and a highfrequency signal between the electrodes 96 and 100.

The low frequency signal is utilized to sense the amplitude of a signalpulse caused by a cell passing through the aperture 92. The highfrequency signal is utilized to obtain the electrical opacity of thesame cell passing through the aperture 92.

The measuring of the electrical opacity of cells was described byWallace H. Coulter and Walter R. Hogg in U.S. Pat. No. 3,502,974 andseveral patents and publications of the assignee, Coulter Electronics,Inc., since that patent. One specific circuit which can be utilizedherein is disclosed in U.S. Pat. No. 4,791,355 entitled PARTICLEANALYZER FOR MEASURING THE RESISTANCE AND REACTANCE OF A PARTICLE, whichis incorporated herein by reference.

The signals generated by the circuit 106 from the sensed cells arecoupled via a DC signal lead 108 and an RF signal lead 110 to acomparator 112 (like the comparator 26). The comparator 112 can hold thesignal generated from the first portion, i.e., those without the WBCsubset subtracted, for a comparison with the results from the secondportion to be described.

The analyzer 86 can include a sheath flow to focus the cells in thesensor 88, in the well known manner. The sheath flow can be provided bya fluidic system 114, coupled to the sensor 88 by a pair of lines 116and 118 in a known manner. The sample reaction mixture can be fed intothe sensor 88 via an introduction tube 120 and can be fed from thesensor 88 via an exit tube 122 into a waste container 124.

While the first portion of the mixture was being analyzed in theanalyzer 86, the second portion is held in the chamber 82, while themixer 72 is cleaned or flushed via a rinse line 126 and exhaustedthrough a waste line 128. Once the chamber 72 is cleaned, the secondportion is fed back into the chamber 72 via a line 130. Like the station30, the WBC subset now is subtracted by adding the WBC microspheres froma station 132 via a line 134, a valve 136 and a chamber line 138.

The WBC microspheres are mixed with the second portion by the mixingmechanism 78. The reaction mixture with the bound WBC microspheres isfed via the line 80, the chamber 82 and the line 84 into the analyzer 86(i.e., the analyzer 34), wherein the second portion is analyzed like thefirst portion and the results then are compared in the comparator 112(i.e., the comparator 26). At least one of the WBC subset cellparameters is changed in the second portion, such as the cell opacity bythe WBC subset bound microspheres to provide the changed results whichthen can be analyzed.

By utilizing magnetic WBC microspheres, then the WBC subset boundthereto can be removed by a magnetic field during and/or after themixing process by a magnetic field or magnet 140. The field can beprovided by electromagnetic means or by the magnet 140 being physicallymoved with respect to the chamber 72 to capture the magnetically boundWBC subset. The second portion without the bound WBC subset then is fedvia the line 80, the chamber 82 and line 84 to the analyzer 86 in themanner previously described to obtain the analysis (like the analyzer34).

The instrument 56 then is prepared to take the next sample for the nextanalysis. The probe 63 can be cleaned by a probe rinse mechanism 142 andthe lines and chamber 72 and 82 can be flushed in a conventional manner.Each analysis of the succeeding sample mixture is obtained in a rapidand automatic fashion. The period between the analysis of succeedingsample mixtures can be on the order of minutes or less.

In operating the analyzer instrument 56, like the analyzer 40, thereaction mixture with the RBC lyse/reactant 46 and the sample 42 ismixed in the chamber 72 along with non-magnetic WBC microspheres fromthe station 132, which bind to one of the WBC subsets. The quench 74 isadded to the reactive mixture which then is fed via the line 80, thechamber 82 and the line 84 to the WBC analyzer 86 for analysis (i.e.,like the analyzer 52).

Alternatively to the utilization of the lyse, in either of the analyzers10 and 40, the sample 12 or 42 can be fed to the mixer 70 via the valve62 without any lyse. In this case, the RBC's can be removed magneticallyby utilizing the microspheres with the RBC specific antibody boundthereto from an RBC microsphere station 144 and fed to the valve 136 viaa line 146 and hence to the chamber 70 via the line 138. Where no lyseis utilized, the bound RBC's are magnetically removed by the magnet 140after mixing in a manner substantially identical to the magneticallybound WBC's described above.

Further, in a second case to promote the speed of the reaction, areaction mixture of the sample with both the RBC lyse and with the RBCmagnetic beads can be utilized. The reaction mixture is mixed, the lyseis quenched and the bound RBC's are magnetically removed and then theWBC's are analyzed as previously described.

Referring now to FIG. 4, another embodiment of a cell populationanalyzing method and apparatus embodying the parent application isdesignated generally by the reference numeral 148. The analyzer 148includes a biological sample 150 which again contains at least a firstset of viable biological cells, such as in or from a whole blood sample.The sample 150 again can include a buffer into which the cells areadded.

The sample 150 is combined via a line 152 with at least one reactant 154via a line 156. The RBC's then are removed as above described by afunctionally designated RBC removing station 158. The reaction mixturewith the RBC's removed is fed via a line 160 into a WBC analyzer 162.The results from the analyzer 162 are fed to a comparator 164 via a line166, providing a three-part WBC differential With results for monocytes(M), lymphocytes (L) and granulocytes (G).

The mixture then is fed to a neutrophil (N) functionally designatedremoval station 168 via a line 170. The N's can be removed from themixture by shifting or changing one parameter, such as opacity, or bymagnetic removal, both as described above. In this example, theparticular N specific antibody utilized is disclosed in U.S. Pat. No.4,931,395.

MONOCLONAL ANTIBODY SPECIFIC TO NEUTROPHILS

The mixture with the N's removed or shifted then is fed to another WBCanalyzer 172 via a line 174. The results of the analyzer 172 are fed tothe comparator 164 via a line 176. The results of the analyzer 172 areutilized to obtain a four-part WBC differential with results again forM's and L's, but now in addition since the N's are shifted or removedresults for eosinophils (E) and basophils (B) are obtained. The twoanalytical results from the analyzers 162 and 172 then can be comparedby the comparator 164 to form a five-part WBC differential.Specifically, subtracting the number of B's and E's from the number ofG's results in the number of the removed N's.

Referring now to FIGS. 5A and 5B, two sets of scattergram results areillustrated obtained from a whole blood sample utilizing a prototypeanalyzing method similar to the analyzer 148. The biological sample 150was a 20 microliter sample of whole blood which was combined with 40microliters of the magnetic microspheres with the RBC specific antibodybound thereto combined with 140 microliters of buffer solution to formthe reactant 154. The reaction mixture was mixed for 15 seconds andplaced in a magnetic field for 10 seconds in the station 158. Themixture with the RBC's removed was analyzed by the analyzer 162 asillustrated in the scattergram of FIG. 5A resulting in counts of L's of45.6 (1), M's of 5.6 (2) and G's of 48.7 (3).

The mixture then is combined in the station 168 with 10 microliters ofmagnetic microspheres with the N specific antibody bound thereto. Themixture is mixed 30 seconds and then placed in a magnetic field for 10seconds. The mixture with the N's then removed was fed to the analyzer176 which resulted in the scattergram of FIG. 5B resulting in counts ofL's of 81.0 (1), M's of 0.6 (2), E's of 11.0 (3) and B's of 1.8 (4). Thecomparator 164 then provides the five-part WBC differential of counts of45.6 L's, 5.6 M's, 41.6 N's, 6.0 E's and 1.2 B's. This corresponds to astandard microscopic five-part WBC differential utilizing Wright stainon the sample on a slide resulting in counts of 44.0 L's, 3.4 M's, 45.0N's, 6.1 E's and 0.4 B's.

FIG. 6 illustrates a further embodiment of a cell population analyzingmethod and apparatus embodying the parent application, designatedgenerally by the reference numeral 178. The analyzer 178 includes abiological sample 180 which again contains at least a first set ofviable biological cells and also can include a buffer.

The sample 180 is combined via a line 182 with a reactant 184 via a line186. Functionally illustrated, a first portion of the mixture is fed viaa line 188 to a functionally designated RBC and N removing station 190.The RBC's and N's are removed or shifted as described before and thefirst portion is fed via a line 192 to a WBC analyzer 194.

This provides a result from the analyzer 194 which is fed via a line 196to a comparator 198. The results includes the above-referenced four-partdifferential including M's, L's, E's and B's.

At the same time, a second portion of the mixture of the sample 180 andthe reactant 184 is fed via a line 200 to a functionally designated RBCremoval station 202. The mixture with the RBC's removed is fed via aline 204 to another WBC analyzer 206. The results of the analyzer 206are fed to the comparator 198 via a line 208. The results of theanalyzer 206 directly include the above-referenced three-part WBCdifferential including M's, L's and G's. The results of the analyzers194 and 206 then are compared by the comparator 198 to provide thefive-part WBC differential.

A specific analyzing instrument embodiment incorporating the method andapparatus of the analyzer 178 is designated generally by the referencenumeral 210 in FIG. 7. Again, only one specific hardware enumeration hasbeen illustrated, but like the analyzing instrument 56, the analyzinginstrument 210 can be implemented in numerous configurations.

The instrument 210 includes an aspirator purging mechanism 212 which iscoupled to a sampling valve 214 via a line 216. The valve 214 caninclude a sample probe 218 to aspirate the biological sample ofinterest, such as the sample 180. A diluent delivery pump 220 is coupledto the valve 214 via a line 222 to provide a diluent for the sample,such as a whole blood sample, when desired. A first portion of themixture then is coupled via a line 224 and a line 226 to a first mixingapparatus 228. At the same time, a second portion of the mixture is fedvia the line 224 and a line 230 to a second mixing apparatus 232.

The mixer 228 (comparable to the station 190) is substantially identicalto the mixer 232 (comparable to the station 202) and will be describedfirst. The mixer 228 includes a mixing chamber 234 into which the firstmixture portion is fed. The mixer 228 includes all of the variousoptions above described and can include a lyse input line 236 for theRBC lyse if desired.

If the lyse is utilized, after mixing as illustrated functionally at238, then the quench is added via a quench line 240. At the same time,the N's are being removed by the addition of the appropriate magnetic ornon-magnetic microspheres with the N specific antibody bound theretofrom a source of microspheres 242 fed to the chamber 234 via a line 244.If magnetic microspheres are utilized for the N's or the RBC's, then amagnet 246 or magnetic field is utilized to remove the magneticallybound cells.

The mixed and quenched (where necessary) mixture then is fed via a line248 through a valve 250 and a line 252 to a WBC analyzer 254 (i.e.,analyzer 194). The analyzer 254 is the same as the analyzer 86 and willnot be described again in such detail. Again, the analyzer 254 includesa sensing chamber 256 with an aperture 258 therein through which themixture and cells pass. A sheath flow fluidic system 260 can be coupledto the chamber 256. The signals generated by the cells are detected byan RF/DC source and sensing circuit 262 whose outputs are fed to acomparator 264, as previously described.

Concurrently, the second mixture portion is fed into a mixing chamber266. In the second portion, only the RBC's are removed (i.e., like thestation 202) and the RBC's can be removed by the RBC lyse fed into thechamber 266 via a line 268. The lyse is mixed with the sample and then aquench is added via a quench line 270. Alternatively the RBC's can beremoved by magnetic microspheres having the RBC specific antibody boundthereto from a microsphere source 272 fed into the chamber 266 via aline 274. The microspheres are mixed, functionally at 276, and then themagnetically bound RBC microspheres are removed by a magnet 278.

The RB removed mixture then is fed via a line 280 to the valve 250 andvia the line 252 to the analyzer 254 to obtain the above-mentionedresults. The mixers 228 and 232 include appropriate respective rinselines 282 and 284 and waste lines 286 and 288 and a probe rinse 290 tocleanse the instrument 210 prior to aspirating the next sample or samplefor analyzing.

FIGS. 8A and 8B illustrate scattergram results obtained from a wholeblood sample utilizing an analyzing method similar to the analyzer 178.In this example, 20 microliters of whole blood form the sample 180,while 40 microliters of magnetic microspheres with the RBC specificantibody bound thereto combined with 140 microliters of buffer solutionform the reactant 184. A portion of the mixture is mixed for 20 secondsin the station 202 and then placed in a magnetic field for 10 seconds.The RBC removed mixture then is analyzed in the analyzer 206 resultingin the scattergram of FIG. 8A which provides a count of L's 29.4 (1),M's 8.1 (2) and G's 62.4 (3).

At the same time, another portion of the same mixture is combined with10 microliters of magnetic microspheres with the N specific antibodybound thereto to remove the RBC's and N's in the station 190. Themixture is mixed for 30 seconds, then placed in a magnetic field for 10seconds. The mixture with the N's and RBC's removed then is analyzed bythe analyzer 194 resulting in the scattergram of FIG. 8B which providesa count of L's 73.5 (1), M's 21.7 (2), E's 3.4 (3) and B's 1.4 (4). Thetwo counts are compared in the comparator 198, resulting in a five-partWBC differential count of L's 29.4, M's 8.0, N's 60.8, E's 1.2 and B's0.6. A microscope comparison again was made resulting in counts of L's29.4, M's 5.0, N's 65.0, E's 1.0 and B's of less than 1.0.

FIGS. 9A and 9B show scattergram results of a five-part WBC differentialexample similar to that of FIGS. 8A and 8B. A 20 microliter sample ofwhole blood was analyzed in the same steps described with respect toFIGS. 8A and 8B resulting in the scattergram of FIG. 9A providing acount of L's 35.4 (1), M's 14.6 (2) and G's 50.0 (3). The scattergram ofFIG. 9B provides a count of L's 66.4 (1), M's 25.0 (2), E's 6.6 (3) andB's 2.0 (4). The resulting five-part WBC differential results in countsof 35.4 L's, 14.6 M's, 45.5 N's, 3.5 E's and 1.1 B's was compared to amicroscope count of 36 L's, 11 M's, 49 N's, 3 E's and 1 B.

FIGS. 10A and 10B show scattergram results of a five-part WBCdifferential again similar to that of FIGS. 8A, 8B and 9A, 9B, however,in this example, lyse was utilized. In this example, 20 microliters ofwhole blood was combined with 80 microliters of buffer and 240microliters of the RBC preferential lyse above referenced. The mixtureis mixed for 6 seconds and then a quench is added. The time period issignificant, because the lyse left unquenched for a period of timegreater than about 10 seconds will start to affect the significantproperties of the WBC's. The mixture with the RBC's removed is analyzedto provide the scattergram of FIG. 10A resulting in counts of L's 25.7(1), M's 9.6 (2) and G's 65.0 (3).

A second portion of the mixture including a second 20 microliter sampleof the whole blood is combined with 120 microliters of buffer and 10microliters of magnetic microspheres with the N specific antibodythereto and mixed for 30 seconds and then placed in a magnetic field for10 seconds. The RBC preferential lyse then is added to the N removedmixture which then is mixed for 6 seconds before it is quenched. Theresulting scattergram FIG. 10B results in percentage counts of L's 74.6(1), M's 21.6 (2), E's 2.9 (3) and B's 0.8 (4). The resulting five-partWBC differential results in percentage counts of L's 25.6, M's 9.6, N's63.5, E's 1.06 and B's 0.3. Again a microscope comparison resulted incounts of L's 29.4, M's 5.0, N's 65.0, E's 1.0 and B's of less than 1.

Another example of scattergram results of a five-part WBC differentialsimilar to that of FIGS. 10A and 10B is illustrated in FIGS. 11A and11B. A sample of whole blood had two samples simultaneously analyzed inthe same steps described with a respect to FIGS. 10A and 10B. Thescattergram of FIG. 11A provides a count of L's 31.9 (1), M's 17.6 (2)and G's 50.4 (3). The scattergram of FIG. 11B provides a count of L's67.1 (1), M's 24.1 (2), E's 7.6 (3) and B's 1.2 (4). The resultingfive-part WBC differential results in counts of 31.9 L's, 11.4 M's, 46.0N's, 3.6 E's and 0.7 B's as compared to a microscope count of 36 L's, 11M's, 49 N's, 3 E's and 1 B's.

A yet still further embodiment of a cell population analyzing method andapparatus embodying the parent application is designated generally bythe reference numeral 292 in FIG. 12. The analyzer 292 includes abiological sample 294, again including at least a first set of viablebiological cells and including a buffer if desired.

The sample 294 is combined via a line 296 with at least one reactant 298via a line 300. In the analyzer 292, the RBC's are removed and the N'sare shifted sequentially or simultaneously in a functionally designatedstation 302. The RBC remove function is designated 304 and the N move orshift portion is designated 306 to indicate that the functions can beperformed simultaneously or sequentially. The RBC's can be removedmagnetically or with lyse or with a combination of the two as previouslydescribed. The N's are removed or shifted by adding microspheres havingan N specific antibody bound thereto to the mixture.

Once the RBC's are removed and the N's are moved or shifted, then theresulting mixture is fed via a line 308 to an analyzer 310. In thiscase, the N's are shifted sufficiently from the patterns of the E's andB's that a five-part WBC differential of M's, L's, E's, B's and N's isdirectly obtained. The functions of the analyzer 292 can be performed oneither of the instruments 56 and 210 or minor variations thereof.

The scattergram results of one example of a direct five-part WBCdifferential in accordance with the analyzer 292 is illustrated in FIG.13. In this example, the biological sample 294 is 20 microliters of awhole blood sample and the reactant 298 is 10 microliters ofnon-magnetic microspheres with the N specific antibody bound theretocombined with 100 microliters of buffer and mixed in the substation 306for 30 seconds. The RBC preferential lyse, 10 microliters thereof, thenis added to the mixture which is mixed in the substation 304 for 6seconds after which the quench is added. The RBC removed and N shiftedmixture then is analyzed by the analyzer 310 resulting in thescattergram of FIG. 13 which provides a direct count of 29.6 L's, 13.6M's, 52.2 N's, 3.4 E's and 1.06 B's as compared to a microscopedetermination of 35 L's, 5 M's, 56 N's, 4 E's and no B's. In thisparticular example, the whole blood sample was also analyzed on ageneral cell counting instrument of Coulter Electronics, Inc., whichresulted in 29 L's, 11.1 M's and 59.9 G's (N's, E's and B's).

Referring now to FIGS. 14 and 15, the embodiments of the presentinvention are illustrated.

The embodiments of the WBC population analyzer methods and apparatus ofthe present invention can be substantially the same as the analyzers 56,148 and 178 illustrated in FIGS. 3, 4 and 6. The analyzers include abiological sample, which contains at least a first set of viablebiological cells (not illustrated), including the white blood cellpopulations.

Applicant has discovered that the multi-part differential procedure ofthe parent application operates efficiently and accurately in a typicalhospital operation, where the blood sample is on the order of 12 hoursold or less. However, when the blood sample is in the range of 18-24hours old or older, the age of the sample causes differences in thelocation of the cell data points on the scattergram. For example anormal blood sample scattergram utilizing the electronics sensingparameters of DC and opacity generate patterns like FIGS. 9A, 10A and11A. The N's are removed from the blood sample resulting in patterns ofL's, M's, E's and B's as shown in FIGS. 9B, 10B and 11B. The parentapplication N removal procedure thus provided the desired five-part WBCdifferential.

However, in an aged blood sample, such as illustrated in FIGS. 14A and15A, the scattergrams are no longer clearly identifiable as L's, G's andM's. The procedure of the parent application produces no or erroneousresults when utilized with the aged samples, FIG. 14A illustrating ablood sample about 26 hours old and FIG. 15A illustrating a blood sampleabout 36 hours old. These aged samples produce patterns which are notreadily identifiable, apparently because of a new population orpopulations, as can be seen by comparing FIGS. 9A, 10A and 11A withFIGS. 14A and 15A.

Referring first to FIG. 14A, Applicant first utilized the electronicsensing parameters of the parent application to obtain the scattergrampattern 350. This pattern 350 was not the same as for a normal(non-aged) blood sample, because as can be seen by comparing, forexample FIGS. 9A, 10A or 11A with FIG. 14A, a new cell grouping 354 nowappears in the aged sample scattergram. Applicant then removed the N'sto obtain a scattergram, 356 illustrated in FIG. 14C, also in accordancewith the parent application. It appeared that some of the cells in thecell grouping 354 were aged N's, which were removed with the other N's.This however did not allow for a proper five-part differential sincesome unidentified cells still remained to form a cell grouping 358. Itappeared that the scattergram 356 included separate L, M, E and Bgroupings, but that still left the grouping 358 unaccounted for. Sincethis technique did not allow a five-part white blood cell differentialto be obtained in accordance with the parent application, Applicant thenutilized the light scatter techniques of the VCS type instrument, abovereferenced to obtain another pattern 352, as illustrated in FIG. 14B.The scattergram 352 also could not be analyzed utilizing the normal VCStechniques, again because of the new undefined population 354.

Applicant then attempted to determine what cell or cells were causingthe scattergram pattern to change with age. To determine what the cellsin the grouping 358 could be, Applicant analyzed the pattern 356. Sinceit appeared that some aged N's were in the pattern 354, Applicantdecided to determine if the pattern 358 was caused by aged E's.Applicant first added the cell count in the pattern 358 to the number ofE's and compared this to a normal (non-aged) five-part differential forthis sample (previously obtained). This indeed corrected the erroneousfive-part differential results, confirming that the remaining cells inthe pattern 358 were aged E's. Applicant then utilized an N and Especific antibody disclosed in U.S. Ser. No. 068,618, entitledMONOCLONAL ANTIBODY SPECIFIC TO A COMMON DETERMINANT SITE OF NEUTROPHILSAND EOSINOPHILS, filed Jun. 13, 1987, which is incorporated herein byreference. Since it appeared that the N's already were removed, theantibody again bound to magnetic microspheres was expected to remove atleast all of the E's. The microspheres removed both all of the Egrouping and the cell grouping 358. This again confirmed that the agedcells in the scattergram 350 were both aged E's and aged N's. The cellsin grouping 358 also could be aged N's that no longer display anaffinity to the N specific antibody, but still display an affinity tothe N and E specific antibody. However, since the final results arecompared back to the zero time results and the number of E's is correct,this suggests the remaining cells are aged E's and not aged N's.

If the cells were aged E's and aged N's as appeared, then the techniqueof the parent application could be applied, with the followingmodifications. The total WBC populations and the L population are firstcounted, including all of the grouping 354. Unlike the scattergram ofthe samples illustrated in FIGS. 9A, 10A and 11A, the G's and M's arenot separately identifiable in the scattergram 350. Therefore, the totalWBC populations are counted as indicated by and included in a block 360.The L's appeared identifiable in both of the scattergrams 350 and 356and hence they are separately counted as indicated by a block 362 inFIG. 14A. The N's then are removed which removes both the N's from theE's area in the scattergram 356 of FIG. 14C as well as the aged N's fromthe grouping 354. The L's, M's, B's and E's then can be counted in thescattergram 356. The L's are correlated with the L's in the block 362.The total WBC populations includes the block or grouping 358. A countfor the removed N's is obtained by comparing the two total WBC counts.The L and total WBC counts can be utilized to correlate the individualM, B and E counts. The E's total counts include the E grouping and theaged E 358 grouping.

Applicant also has determined that the errors caused by the aging of thesample cannot be precisely predicted as to time of occurrence. However,the errors appear to occur on a one cell to one cell basis and hence thetechnique of the present invention can be utilized at any time. In thecase of a normal pattern such as shown in FIGS. 9A, 10A and 10B, thecount for the groupings 354 and 358 will be zero. The technique thusensures a correct five-part differential, whether or not there are anyaged cells.

To confirm the results of the technique of the present invention,samples were tested utilizing the VCS instrument at zero hours agingwhich are compared to some aged 24 hour samples utilizing the presentinvention. The results compare very well as illustrated by Table I.

                  TABLE I                                                         ______________________________________                                        Patient:        Patient:                                                              VCS     24 HR             VCS  24 HR                                  ______________________________________                                        LYMPH:  34.13   36.0    LYMPH:    31.6 29.8                                   MONO:   9.67    9.0     MONO:     11.7 12.0                                   NEUTRO: 52.68   51.4    NEUTRO:   48.07                                                                              49.6                                   EOS:    3.06    2.9     EOS:      7.4  7.2                                    BASO:   0.46    0.5     BASO:     1.2  1.5                                    ______________________________________                                        Patient:        Patient:                                                              VCS     24 HR             VCS  24 HR                                  ______________________________________                                        LYMPH:  18.76   19.0    LYMPH:    27.63                                                                              27.4                                   MONO:   8.6     7.8     MONO:     6.5  4.9                                    NEUTRO: 69.18   69.7    NEUTRO:   49.5 50.1                                   EOS:    2.81    3.2     EOS:      15.9 16.4                                   BASO:   0.7     0.2     BASO:     0.5  1.2                                    ______________________________________                                        Patient:        Patient:                                                              VCS     24 HR             VCS  24 HR                                  ______________________________________                                        LYMPH:  26.6    27.4    LYMPH:    33.24                                                                              33.5                                   MONO:   10.67   9.8     MONO:     9.18 7.7                                    NEUTRO: 59.8    60.6    NEUTRO:   49.73                                                                              51.3                                   EOS:    2.86    3.3     EOS:      6.76 6.6                                    BASO:   0.19    0.7     BASO:     1.09 1.0                                    ______________________________________                                        Patient:        Patient:                                                              VCS     24 HR             VCS  24 HR                                  ______________________________________                                        LYMPH:  28.2    27.4    LYMPH:     40.62                                                                             40.1                                   MONO:   9.3     8.9     MONO:     6.77 3.6                                    NEUTRO: 57.80   59.1    NEUTRO:   50.76                                                                              53.0                                   EOS:    3.9     3.9     EOS:      1.04 2.6                                    BASO:   0.6     0.8     BASO:     0.81 0.9                                    ______________________________________                                        Patient:        Patient:                                                              VCS     24 HR             VCS  24 HR                                  ______________________________________                                        LYMPH:  35.21   33.3    LYMPH:    38.41                                                                              36.2                                   MONO    8.91    8.9     MONO:     13.09                                                                              15.7                                   NEUTRO: 47.65   48.9    NEUTRO:   39.49                                                                              38.5                                   EOS:    7.86    8.9     EOS:      7.03 5.8                                    BASO    0.38    0.8     BASO:     1.4  1.7                                    ______________________________________                                        Patient:        Patient:                                                              VCS     24 HR             VCS  24 HR                                  ______________________________________                                        LYMPH:  24.20   23.6    LYMPH:    32.69                                                                              34.6                                   MONO:   9.27    7.4     MONO:     10.37                                                                              7.1                                    NEUTRO: 62.23   64.2    NEUTRO:   50.32                                                                              51.6                                   EOS:    3.33    3.6     EOS:      5.82 6.1                                    BASO:   0.98    1.3     BASO:     0.79 0.6                                    ______________________________________                                        Patient:        Patient:                                                              VCS     24 HR             VCS  24 HR                                  ______________________________________                                        LYMPH:  34.35   37.8    LYMPH:    33.62                                                                              34.0                                   MONO:   9.37    7.4     MONO:     7.44 7.2                                    NEUTRO: 54.73   52.6    NEUTRO:   56.33                                                                              54.8                                   EOS:    0.95    1.3     EOS:      1.87 2.8                                    BASO:   0.58    0.9     BASO:     0.75 1.1                                    ______________________________________                                        Patient:        Patient:                                                              VCS     24 HR             VCS  24 HR                                  ______________________________________                                        LYMPH:  30.41   29.3    LYMPH:    40.53                                                                              38.7                                   MONO:   6.65    4.5     MONO:     12.35                                                                              10.7                                   NEUTRO: 58.86   59.7    NEUTRO:   45.57                                                                              46.7                                   EOS:    3.39    4.8     EOS:      1.19 3.0                                    BASO:   0.80    1.6     BASO:     0.31 0.3                                    ______________________________________                                    

The 24 hour sample results in Table I all sere obtained utilizingelectronic RF and DC sensing parameters. To confirm that the presentinvention also can be utilized with light scatter, such as in the VCSinstrument, one set of 24 hour sample data was obtained as illustratedin Table II.

                  TABLE II                                                        ______________________________________                                        Patient:                                                                                    VCS  24 HR VCS                                                  ______________________________________                                        LYMPH:          15.6   15.5                                                   MONO            8.2    6.9                                                    NEUTRO:         72.2   74.5                                                   EOS:            4.1    3.1                                                    BASO:           --     --                                                     ______________________________________                                    

The techniques of the present invention also were applied to 36 hoursaged samples as illustrated in FIGS. 15A-C. Another scattergram 364produced by electronic sensing is illustrated in FIG. 15A, while a VCSscattergram 366 is illustrated in FIG. 15B. Again, the total white bloodcell populations are counted as indicated by a block or grouping 368 andthe L's also are counted as indicated by a block or grouping 370. TheN's then are removed as before, including the aged N's resulting in ascattergram 372, which again includes the L's, M's, E's, B's and agedE's in a grouping 374.

Results from the 36 hour aged samples can be compared with the VCS zeroage data in Table III.

                  TABLE III                                                       ______________________________________                                        Patient:        Patient:                                                              VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  46.6    44.4    LYMPH:    31.7 32.8                                   MONO    11.1    10.2    MONO:     11.3 10.4                                   NEUTRO: 37.1    39.6    NEUTRO:   50.7 49.9                                   EOS:    5.2     5.0     EOS:      5.0  6.6                                    BASO:   0.04    0.8     BASO:     1.2  0.3                                    ______________________________________                                        Patient:        Patient:                                                              VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  30.1    28.6    LYMPH:    29.9 28.1                                   MONO:   9.6     7.5     MONO:     13.6 14.8                                   NEUTRO: 55.3    58.8    NEUTRO:   49.2 49.1                                   EOS:    4.2     4.5     EOS:      5.5  5.1                                    BASO:   0.7     0.6     BASO:     1.7  2.9                                    ______________________________________                                        Patient:        Patient:                                                              VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  39.5    36.4    LYMPH:    29.9 25.3                                   MONO:   9.9     6.2     MONO:     7.7  6.2                                    NEUTRO: 45.2    51.6    NEUTRO:   57.3 61.8                                   EOS:    3.7     5.2     EOS:      4.9  5.8                                    BASO:   1.7     0.6     BASO:     0.2  0.9                                    ______________________________________                                        Patient:        Patient:                                                              VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  41.3     41.3   LYMPH:    30.1 27.9                                   MONO:   6.8     7.9     MONO:     8.4  6.6                                    NEUTRO: 44.3    43.0    NEUTRO:   54.5 56.9                                   EOS:    6.4     6.6     EOS:      6.5  8.2                                    BASO:   1.2     1.2     BASO:     0.5  0.4                                    ______________________________________                                        Patient:        Patient:                                                              VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  36.6    32.9    LYMPH:    31.2 30.0                                   MONO:   7.1     5.7     MONO:     6.7  5.6                                    NEUTRO: 52.1    55.0    NEUTRO:   57.6 57.1                                   EOS:    3.5     5.2     EOS:      3.9  6.3                                    BASO:   1.4     1.2     BASO:     0.5  0.1                                    ______________________________________                                        Patient:        Patient:                                                              VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  38.6    35.2    LYMPH:    35.7 31.5                                   MONO:   8.4     7.3     MONO:     13.7 13.5                                   NEUTRO: 49.5    52.5    NEUTRO:   44.1 46.3                                   EOS:    3.2     4.4     EOS:      5.4  5.7                                    BASO:   0.3     0.6     BASO:     1.0  2.0                                    ______________________________________                                        Patient:        Patient:                                                              VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  34.5    31.6    LYMPH:    37.0 33.2                                   MONO:   7.6     6.3     MONO:     7.5  6.1                                    NEUTRO: 56.4    59.6    NEUTRO:   50.1 54.1                                   EOS:    1.1     2.2     EOS:      4.8  4.8                                    BASO:   0.4     0.3     BASO:     0.5  1.8                                    ______________________________________                                        Patient:        Patient:                                                              VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  31.8    30.3    LYMPH:    41.5 37.5                                   MONO:   11.3    8.8     MONO:     5.9  4.2                                    NEUTRO: 50.4    54.2    NEUTRO:   45.2 49.5                                   EOS:    5.0     5.7     EOS:      6.9  8.0                                    BASO:   1.4     1.0     BASO:     0.7  0.9                                    ______________________________________                                        Patient:        Patient:                                                              VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  39.5    36.9    LYMPH:    41.4 40.6                                   MONO    8.9     6.8     MONO:     7.6  7.4                                    NEUTRO: 49.8    52.9    NEUTRO:   47.1 47.4                                   EOS:    1.5     2.8     EOS:      3.4  3.7                                    BASO:   0.3     0.6     BASO:     0.5  0.7                                    ______________________________________                                        Patient         Patiient:                                                             VCS     36 HR             VCS  36 HR                                  ______________________________________                                        LYMPH:  25.9    25.5    LYMPH:    28.7 27.7                                   MONO:   6.4     5.5     MONO:     9.8  8.4                                    NEUTRO: 62.8    62.2    NEUTRO:   59.2 61.1                                   EOS:    3.8     5.6     EOS:      1.7  1.9                                    BASO:   1.0     1.2     BASO:     0.5  0.9                                    ______________________________________                                    

The magnetic microspheres utilized can be of any suitable type and forexample, are polystyrene magnetic microspheres of 0.7 micron diameter,with a weight to volume of 10% solids, sold by Bangs Laboratories ofCarmel, Ind. The non-magnetic microspheres again can be of any suitabletype and for example, are surfactant free sulfated polystyrene latexmicrospheres of 2.17 micron diameter with a weight to volume of 8%solids, sold as IDC microspheres by Interfacial Dynamics of Portland,Oreg. Although these specific microspheres are utilized for examplepurposes, other types and sizes of microspheres from other conventionalsources also can be utilized.

In general, for shifting, it is preferable to utilize microspheres of adiameter substantially less than the diameter of the cells, since aplurality of the microspheres should bind to each cell. Typically,microspheres of diameters from 0.65 to 3.0 microns are utilized toensure that the instrument does not sense and count the freemicrospheres themselves as an indicated cell. In general, the cellantigen density also affects the size of microspheres utilized, since alow density (binding a few microspheres) would require largermicrospheres and a high density (binding numerous microspheres) wouldallow use of smaller microspheres. If the microspheres are too large thesystem can count the free microspheres as cells to generate aninaccurate count and hence an inaccurate analysis. Further, very largemicrospheres could clog or block the sensing apertures, since aplurality of cells could be bound thereto, resulting in a largemicrosphere and cell mass. Typically, non-magnetic microspheres are lessexpensive and hence are utilized for shifting purposes, althoughmagnetic microspheres also can be utilized to cause the sensed cellshift.

To eliminate cells magnetically, only magnetic microspheres can beutilized. In this application, the microsphere size is less relevant,since the cells are to be eliminated from the sample before sensing. Themicrospheres should be of sufficient means to quickly and easily beremoved in the magnetic field. In this case, 0.65 to 4.5 micron diametermicrospheres work well. Further, since the cells only are to beeliminated, 10 or greater micron diameter microspheres could beutilized. A plurality of cells could be bound to each microsphere, butsince no counting will take place, this does not interfere with theoperation of the instrument.

I claim:
 1. A method of obtaining a not otherwise obtainable multi-partwhite blood cell population differential from at least a portion of awhole blood sample having at least white blood cell populations thereinfor identification and/or enumeration and/or study,comprising:electronically counting at least the total white blood cellpopulations and the lymphocyte population; removing the neutrophilpopulation including the aged neutrophil population contribution fromsaid white blood cell populations by providing microspheres having aneutrophil specific monoclonal antibody bonded thereto and mixing saidmicrospheres with said sample to bind to said neutrophil populations andremoving said microspheres with said neutrophil populations boundedthereto from said sample and in so doing allowing for the electronicsensing of eosinophils and basophils; electronically counting at leastsaid remaining white blood cell populations of monocytes, lymphocytes,eosinophils, aged eosinophils and basophils; and comparing said twocounts to derive a count of said white blood cell populations ofneutrophils and aged neutrophils and thereby obtaining at least afive-part white blood cell differential.
 2. The method as defined inclaim 1 wherein said whole blood sample includes a red blood cellpopulation and removing said red blood cell population from said samplewithout adversely affecting relevant qualities and/or quantities of saidwhite blood cell populations.
 3. The method as defined in claim 2wherein removal of said red blood cell population includes providingmicrospheres having a red blood cell specific monoclonal antibody bondedthereto and mixing said microspheres with said whole blood sample tobind to said red blood cell population; andremoving said microsphereswith said bound red blood cells from said whole blood sample.
 4. Themethod as defined in claim 3 including providing magnetic microspheresand a magnetic field and removing said microspheres by removing said redblood cells while attracting said magnetic microspheres within saidmagnetic field.
 5. The method as defined in claim 3 including rapidlymixing said microspheres with said whole blood sample to bind said redblood cell population to said microspheres in less than sixty seconds.6. The method as defined in claim 2 wherein removing said red blood cellpopulation includes providing microspheres having a red blood cellspecific monoclonal antibody bonded thereto and mixing said microsphereswith said whole blood sample to bind to said red blood cell populationand providing a red blood cell lyse with said microspheres to eliminatea portion of said red blood cell population to decrease the number ofmicrospheres necessary to remove said red blood cell population;andremoving said microspheres with said red blood cells bound theretofrom said whole blood sample.
 7. The method as defined in claim 6including providing magnetic microspheres and a magnetic field andremoving said microspheres by removing said red blood cells whileattracting said magnetic microspheres within said magnetic field.
 8. Themethod as defined in claim 6 including rapidly mixing said microsphereswith said whole blood sample to bind said red blood cell population tosaid microspheres in less than sixty seconds and to provide said lysingaction.
 9. The method as defined in claim 2 wherein removing said redblood cell population includes providing a red blood cell lyse tosubstantially eliminate said red blood cell population.
 10. The methodas defined in claim 1 including providing magnetic microspheres and amagnetic field and removing said microspheres by removing saidneutrophil populations while attracting said magnetic microsphereswithin said magnetic field.
 11. The method as defined in claim 1including rapidly mixing said microspheres with said sample to bind saidneutrophil populations to said microspheres in less than sixty seconds.12. The method as defined in claim 1 wherein comparing said two countsincludes comparing the two lymphocyte counts to correlate the populationcounts.
 13. A method of obtaining a not otherwise obtainable multi-partwhite blood cell population differential from at least a portion of awhole blood sample having at least white blood cell populations thereinfor identification and/or enumeration and/or study,comprising:electronically counting at least the total white blood cellpopulations and the lymphocyte population; removing the neutrophilpopulation including the aged neutrophil population contribution fromsaid white blood cell populations from a second portion of said samplewithout adversely affecting the relevant qualities and/or quantities ofsaid remaining white blood cell populations by providing microsphereshaving a neutrophil specific monoclonal antibody bonded thereto andmixing said microspheres with said sample to bind to said neutrophilpopulations and removing said microspheres with said neutrophilpopulations bound thereto from said sample and in so doing allowing forthe electronic sensing of eosinophils and basophils; electronicallycounting at least said remaining white blood cell populations ofmonocytes, lymphocytes, eosinophils, aged eosinophils and basophils insaid second portion; and comparing said two counts from said first andsecond portions to derive a count of said white blood cell populationsof neutrophils and aged neutrophils and thereby obtaining at least afive-part white blood cell differential.
 14. The method as defined inclaim 13 wherein said whole blood sample includes a red blood cellpopulation and removing said red blood cell population from said samplewithout adversely affecting relevant qualities and/or quantities of saidwhite blood cell populations prior to the counting of said first portionand also removing said red blood cell population from said secondportion prior to the counting thereof.
 15. The method as defined inclaim 14 wherein removing said red blood cell population from at leastone of said portions includes providing microspheres having a red bloodcell specific monoclonal antibody bonded thereto and mixing saidmicrospheres with said whole blood sample to bind to said red blood cellpopulation; andremoving said microspheres with said red blood cellsbound thereto from said whole blood sample.
 16. The method as defined inclaim 15 including providing magnetic microspheres and a magnetic fieldand removing said microspheres by removing said red blood cells whileattracting said magnetic microspheres within said magnetic field. 17.The method as defined in claim 15 including rapidly mixing saidmicrospheres with said whole blood sample to bind to said red blood cellpopulation to said microspheres in less than sixty seconds.
 18. Themethod as defined in claim 14 wherein removing said red blood cellpopulation from at least one of said portions includes providingmicrospheres having a red blood cell specific monoclonal antibody bondedthereto and mixing said microspheres with said whole blood sample tobind to said red blood cell population and providing a red blood celllyse with said microspheres to eliminate a portion of said red bloodcell populations to decrease the number of microspheres necessary toremove said red blood cell populations; andremoving said microsphereswith said red blood cells bound thereto from said whole blood sample.19. The method as defined in claim 18 including providing magneticmicrospheres and a magnetic field and removing said microspheres byremoving said red blood cells while attracting said magneticmicrospheres within said magnetic field.
 20. The method as defined inclaim 18 including rapidly mixing said microspheres with said wholeblood sample portion to bind said red blood cell population to saidmicrospheres in less than sixty seconds and to provide said lysingaction.
 21. The method as defined in claim 14 wherein removing said redblood cell population from at least one of said portions includesproviding a red blood cell lyse to substantially eliminate said redblood cell population.
 22. The method as defined in claim 13 includingproviding magnetic microspheres and a magnetic field and removing saidmicrospheres by removing said neutrophil populations while attractingsaid magnetic microspheres within said magnetic field.
 23. The method asdefined in claim 13 including rapidly mixing said microspheres with saidwhole blood sample to bind said neutrophil populations to saidmicrospheres in less than sixty seconds.
 24. The method as defined inclaim 13 wherein comparing said two counts includes comparing the twolymphocyte counts to correlate the population counts.